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Une des clés pour comprendre les mécanismes d’action biologiques des molécules naturelles et thérapeutiques est leur faculté à incorporer ou traverser les membranes lipidiques. Parce que les méthodes expérimentales sont parfois couteuses et répondent partiellement aux questions posés par les interactions composé-membrane, la modélisation moléculaire est devenue une sérieuse alternative. Les simulations de dynamique moléculaire ont ouvert de nombreuses perspectives ces dernières années en offrant la possibilité de décrire ces interactions intermoléculaires au niveau atomique. À l’aide de ces simulations, nous avons évalué la capacité de plusieurs composés (polyphénols, vitamines E et C, plantazolicine et carprofènes) à s’incorporer dans les membranes. Ces molécules ont été choisies pour leurs activités biologiques diverses, à savoir (i) activité antioxydante, précisément inhibition de la peroxydation lipidique, (ii) activité antibiotique et possibilité de former un pore transmembranaire, et (iii) inhibition d’enzymes impliquées dans la maladie d’Alzheimer. Leurs positions et orientations ainsi que leur capacité à s’accumuler ou à traverser les membranes ont été évaluées pour comprendre leurs mécanismes d’action.Dans le but d’utiliser les simulations de dynamique moléculaire en drug design, l’accent a été mis sur la précision des calculs, qui dépend de la qualité sous-jacente du modèle utilisé. En corrélant données expérimentales et théoriques, la méthodologie de nos modèles a été systématiquement revisitée. Le choix du champ de force, les paramètres des composés étudiés ainsi que la composition de la membrane sont en particulier apparus comme d’importants facteurs dans la description des interactions entre les molécules naturelles et thérapeutiques et les membranes. Des mélanges de lipides contenant du cholestérol ont notamment été utilisés et ont montré un impact significatif sur les résultats obtenus. / One of the key lockers to understand mechanisms of biological action of drugs and natural compounds is their capacity to incorporate/cross lipid bilayer membranes. In the light of demanding experimental techniques, in silico molecular modelling has become a powerful alternative to tackle these issues. In the past few years, molecular dynamics (MD) has opened many perspectives, providing an atomistic description of the related intermolecular interactions. Using MD simulations, we have explored the capacity of several compounds (polyphenols, vitamins E and C, plantazolicin, carprofens) to incorporate lipid bilayer membranes. The different compounds were chosen according to their different biological functions, namely (i) antioxidant activity against lipid peroxidation, (ii) antimicrobial activity with the possibility of trans-membrane pore formation, and (iii) inhibition of enzymes involved in Alzheimer’s disease. In order to rationalize their mechanisms of action, their position and orientation in membranes as well as their capacity to accumulate or permeate lipid bilayers were assessed. Having in mind a predictive purpose in drug design for MD simulations, the accuracy of the results relies on the quality of the in silico membrane models. By ensuring relationships between experimental and theoretical data, methodological improvements have been proposed. In particular, force field selection, xenobiotic parameterization and bilayer constitution emerged as crucial factors to appropriately depict drug-membrane interactions. For the latter issue, lipid mixtures e.g., including cholesterol have been developed.

L’appréhension des mécanismes d’action biologiques des protéines membranaires nécessite de comprendre les interactions des xénobiotiques avec ces protéines et avec les membranes lipidiques. Les méthodes expérimentales sont parfois coûteuses et ne permettent d’obtenir que des informations partielles sur les interactions xénobiotiques-membrane-protéine. La modélisation moléculaire est une sérieuse alternative. Les simulations de dynamique moléculaire et de dynamique biaisées ont ouvert de nombreuses perspectives en permettant de décrire ces interactions moléculaires à l’échelle atomique. Grâce à des simulations de dynamique moléculaire, nous avons été capables de construire un modèle de transporteur humain ABC : ABCC4/MRP4. Cette protéine a été choisie pour sa présence dans le rein, notamment, et son importance clinique. Nous avons évalué l’influence du cholestérol sur cette protéine. L’étude de domaines spécifiques et l’impact d’un polymorphisme a été reliée à l’activité de transport de cette protéine. Nous avons également étudié l’interaction de xénobiotiques avec ce transporteur humain. Le cycle de transport des transporteurs ABC a été examiné afin de comprendre leur fonctionnement. L’incorporation de cholestérol a montré un impact significatif sur la protéine humaine ABCC4/MRP4 et sur les xénobiotiques étudiés. L’importance de domaines constituant la protéine ABCC4/MRP4 ainsi que l’importance de résidus individuels a clairement été prouvée. Nous avons également pu observer des intermédiaires du cycle de transport d’un transporteur ABC conjointement avec des changements structuraux. / Understanding the biological mechanisms of action of membrane proteins requires the comprehension of the interactions of xenobiotics with these proteins and with lipid membranes. Experimental methods are often demanding and only partially respond to xenobiotic-membrane-protein interactions. In silico molecular modeling is a serious alternative to tackle these issues. Molecular dynamics (MD) and biased dynamics simulations have opened many perspectives by providing an atomistic description of these intermolecular interactions. Using MD simulations, we built a model of the human ABC ABCC4/MRP4 transporter. We explored the influence of cholesterol on this protein as well as the impact of a polymorphism known to shut down the transport activity of this protein. We also studied the interaction of xenobiotics with this human transporter. The transport cycle of the ABC transporters was investigated in an attempt to better understand how it works.Interactions between lipid membranes and xenobiotics were explored by examining their ability to incorporate lipid membranes. Lipid mixtures with cholesterol showed a significant impact on the human protein ABCC4/MRP4 and on the xenobiotics studied. The importance of regions, domains constituting the ABCC4/MRP4 protein as well as the importance of specific residues has been clearly demonstrated. We also observed intermediates in the transport cycle of an ABC transporter in conjunction with structural changes occurring during this cycle.

Amyloid-proteins are among the most actively researched biological topics today, because they have been associated with many serious human diseases, such as Alzheimer's disease and type II diabetes. In particular the deposition of protein aggregates on cell membranes has been suspected as the causes of the diseases, although the proof is still elusive. Studying the interactions of amyloid-forming peptides with lipid-bilayer membranes may clarify the pathway of the β-aggregate formation and provide new insights into the amyloid hypothesis of diseases. In this thesis, I investigate how three peptides, penetratin, amylin, and LL-37, interact with lipid membranes by using several techniques well-developed in our lab. In the study of penetratin interacting with lipid membranes, we were able to clarify the energy pathway of amyloid formation mediated by membrane-binding. This provides the sole experimental proof for the Jarrett-Lansbury theory of β- amyloid formation. Our investigation on amylin-membrane interaction clarifies how amylin in different forms damage bilayer membranes. Between penetratin and amylin we have clarified the complicated pattern of interactions between amyloid-forming peptides and lipid bilayers. The third peptide LL-37 studied in my thesis turned out to a pore forming peptide. I found the mistake made by previous investigators in several different laboratories that made them erroneously conclude that LL-37 was not a pore forming peptide. The results of these three peptides show that methods we used are a comprehensive set of tools that can reveal a broad range of peptide properties. Both the formation of amyloid aggregates and formation of membrane pores can be explained by a two-state model proposed by Huang describing peptide-membrane interactions. For LL-37, the second state is a pore in membrane. But for penetratin and amylin the second state is an aggregation in the β form. We found that β-aggregates have low affinity within a lipid bilayer, and therefore exit from the bilayer structure. However, this exit process extracts lipid molecules from the bilayer and incorporates them in the peptide aggregates. We suggest that this is the molecular process of how amylin might damage of the membranes of β-cells.

abstract: Biological membranes are critical to cell sustainability by selectively permeating polar molecules into the intracellular space and providing protection to the interior organelles. Biomimetic membranes (model cell membranes) are often used to fundamentally study the lipid bilayer backbone structure of the biological membrane. Lipid bilayer membranes are often supported using inorganic materials in an effort to improve membrane stability and for application to novel biosensing platforms. Published literature has shown that a variety of dense inorganic materials with various surface properties have been investigated for the study of biomimetic membranes. However, literature does not adequately address the effect of porous materials or supports with varying macroscopic geometries on lipid bilayer membrane behavior. The objective of this dissertation is to present a fundamental study on the synthesis of lipid bilayer membranes supported by novel inorganic supports in an effort to expand the number of available supports for biosensing technology. There are two fundamental areas covered including: (1) synthesis of lipid bilayer membranes on porous inorganic materials and (2) synthesis and characterization of cylindrically supported lipid bilayer membranes. The lipid bilayer membrane formation behavior on various porous supports was studied via direct mass adsorption using a quartz crystal microbalance. Experimental results demonstrate significantly different membrane formation behaviors on the porous inorganic supports. A lipid bilayer membrane structure was formed only on SiO2 based surfaces (dense SiO2 and silicalite, basic conditions) and gamma-alumina (acidic conditions). Vesicle monolayer adsorption was observed on gamma-alumina (basic conditions), and yttria stabilized zirconia (YSZ) of varying roughness. Parameters such as buffer pH, surface chemistry and surface roughness were found to have a significant impact on the vesicle adsorption kinetics. Experimental and modeling work was conducted to study formation and characterization of cylindrically supported lipid bilayer membranes. A novel sensing technique (long-period fiber grating refractometry) was utilized to measure the formation mechanism of lipid bilayer membranes on an optical fiber. It was found that the membrane formation kinetics on the fiber was similar to its planar SiO2 counterpart. Fluorescence measurements verified membrane transport behavior and found that characterization artifacts affected the measured transport behavior. / Dissertation/Thesis / Ph.D. Chemical Engineering 2011

This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biology. / Die vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen.

Le mécanisme d‘interaction d‘un composé xénobiotique avec la membrane est un des facteurs clés qui influence son mécanisme d‘action biologique et donc son action thérapeutique pour un principe actif. Une analyse précise des interactions intermoléculaires à l‘échelle atomique peut être obtenue par dynamique moléculaire, une méthode qui apparait plus que jamais comme une alternative élégante aux techniques expérimentales. Les simulations de dynamique moléculaire permettent d‘évaluer ces interactions avec une résolution temporelle et spatiale difficiles à atteindre avec les méthodes expérimentales. Ces informations constituent une pierre angulaire de la compréhension des mécanismes d‘action des xénobiotiques . Les résultats obtenus corrèlent généralement bien avec les données expérimentales. Dans ce travail théorique, nous avons utilisé des dynamiques moléculaires non -biaisées et biaisées (z-Contraint). Nous avons étudié les modes d‘insertion (positionnement et orientation), les coefficients de partition, et la capacité de différents xénobiotiques à traverser la bicouche lipidique (perméation passive). Plusieurs composés de différentes familles thérapeutiques ont été étudiés (antiviraux, immunosuppresseurs et antioxydants), tous étant utilisés en transplantation d‘organes ; les antioxydants sont étudiés en tant que protecteurs d‘organe contre les phénomènes d‘ischémie -reperfusion. Pour la perméation passive, les profils d‘ énergies, les coefficients de diffusion locaux et la résistance à la traversée ont été calculés pour finalement obtenir des coefficients globaux de perméabilité. Nous avons montré que ces techniques de calcul donnent une description qualitative du processus d‘insertion/perméation, montrant notamment le rôle de différentes propriétés physiques (ex., polarité, charge). Des résultats remarquables ont été obtenus pour les larges molécules. Malgré la taille, ces mol cules peuvent s‘ insérer dans la bicouche lipidique relativement facilement (faibles barrières énergétiques). Par contre, leur diffusion dans les différentes régions de la membrane peut augmenter d‘une manière signifiante. Ce travail donne une confiance accrue dans les méthodes de dynamique moléculaire pour devenir prédictive dans les années avenirs, et aide de façon concrète les pharmacologues dans la recherche de nouvelles stratégies thérapeutiques. / The mechanism of interaction between drugs or any xenobiotic and membrane is one of thekey factors that affect its biological of action, and so its therapeutic activity. A thoroughrationalization of the relationship between the intrinsic properties of the xenobiotics and theirmechanism of interaction with membranes can now be assessed with atomistic details.Molecular dynamics (MD) is a powerful research tool to study xenobiotics-membraneinteractions, which can access time and space scales that are not simultaneously accessibleby experimental methods. Semi-quantitative molecular and thermodynamic descriptions ofthese interactions can be provided using in silico model of lipid bilayers, often in agreementwith experimental measurements.The main goal of our investigation consisted to get in depth insight into the mechanisms ofinteraction/partitioning/insertion/crossing with/in/into/through membrane and drug deliveryusing MD. In this thesis, we have focused on both drugs used in renal transplantation (e.g.,antivirals, immunosuppressants) and antioxidants, which can also be used to protect organsalong the transplantation processes. We have provided a series of clues showing that MDsimulations can tackle the delicate process of drug passive permeation.Both, unbiased and biased MD (z-constraint) simulations have been used to elucidate thexenobiotics-membrane interactions (i.e., positioning and orientation) and to evaluate crossingenergies, diffusion coefficients, and permeability coefficients. These findings led us to drawqualitative structure-permeability relationships (SPR). We have carefully analyzed how thechemical and physical properties of xenobiotics affect the mechanism of interactions andthus permeability. The robustness of these MD-based methodologies has been determinedto qualitatively predict these pharmacological parameters. Hydrophobic compounds showeda favorable partitioning into the lipid bilayer and relatively low Gibbs energy of crossing thecenter of membrane (ΔGcross). Hydrophilic or charged compounds showed partitioning closeto membrane surface, in interaction with the polar head groups and water molecules; this hasbeen shown to dramatically increase ΔGcross. Amphiphilic compounds are intermediatecompounds in terms of membrane insertion/positioning/crossing. It clearly appears that theyshould be analyzed case by case, an analysis for which MD simulations could be particularlysupportive. Also the influence of size at predicting permeation has been studied (i.e.,relatively large drugs were tested). The molecular size has shown no significant influence onΔGcross whereas diffusion coefficients were significantly affected, depending on themembrane regions.

This work reports on the successful formation of supported multicomponent lipid bilayer membranes (sLBMs) from natural occurring lipids as well as synthetic lipids on a set of polymer cushions consisting of alternating maleic acid copolymers. Maleic acid copolymers provide a versatile platform to adjust the physico-chemical behaviour by the choice of the comonomer unit. The formation of sLBMs was triggered by a transient reduction of the electrostatic repulsion between the polymer cushions and the lipid vesicles by lowering the solutions pH to 4. Upon formation the stability of sLBMs was not affected by subsequent variations of the environmental pH to 7.2. Even drastic changes in the environmental pH (between pH 2 and pH 9) did not lead to delamination and proved the stability of the polymer sLBM. The degree of hydrophilicity and swelling of the anionic polymer cushions was found to determine both the kinetics of the membrane formation and the mobility of the lipid bilayer with lipid diffusion coefficients in the range from 0.26 to 2.6 µm2 s-1. An increase in cushion hydrophilicity correlated with a strong increase in the diffusion coefficient of the lipids. This trend was found to correlate with the kinetics of bilayer formation in the process of vesicle spreading. The observations strongly support the important role of the support’s polarity for the fluidity of the sLBM, which is probably related to the presence of a water layer between support and bilayer. The investigated polymer cushions are considered to open new options for the in situ modulation of lipid bilayer membranes characteristics to match the requirements for the successful integration of functional transmembrane proteins (TMPs). As each cushion exhibits different physico-chemical properties, the resulting behaviour of the sLBMs and TMPs could be exactly adjusted to the specific requirements of biological samples. This is exemplarily shown by the integration of the TMP beta amyloid precursor protein cleaving enzyme (BACE). Integrated BACE was observed to be mobile on all polymer cushions. On the contrary, no lateral mobility of BACE was found in solid sLBM. Furthermore, the activity of integrated BACE was analysed by the cleavage of an amyloid precursor protein analogue. Remarkably, the polymer cushions did not only enhance the mobility but were also found to increase the activity of BACE by a factor of 1.5 to 2.5 in comparison to solid sLBM. From the obtained results it is obvious that even small cytoplasmic domains of transmembrane proteins might not be preserved upon the integration in silica sLBM. The observed beneficial effects of the utilised polymer cushions on the mobility and activity of transmembrane proteins motivate further studies to clarify the general applicability of the polymer platform. Altogether, this polymer platform provides valuable options to form sLBM with varying characteristics to reconstitute transmembrane proteins for a wide range of possible future applications in biology. / Die vorliegende Arbeit beschreibt die Bildung von polymer unterstützten Lipiddoppelschichten zur Integration von transmembranen Proteinen. Das Polymerkissensystem besteht aus alternierenden Maleinsäurecopolymeren. Lipiddoppelschichten wurden durch die Steuerung der elektrostatischen Repulsion erzeugt: die Verringerung des pH-Wertes auf 4 wurde eine Erhöhung der adsorbierten Vesikelmenge auf den Polymeroberflächen induziert. Nach der erfolgten Bildung der Lipiddoppelschichten kann der pH-Wert beliebig variiert werden, ohne dass die Stabilität der Lipiddoppelschichten beeinflusst wird. Auch drastische Veränderungen des pH-Milieus (pH 2 - pH 9) führten zu keinen Veränderungen in der Membranintegrität. Der Grad der Hydrophilie und der Quellung der anionischen Polymerschichten beeinflusst sowohl die Bildung der Modellmembranen als auch die Mobilität der integrierten Lipidmoleküle. Dabei reichen die erzielten Lipiddiffusionskoeffizienten von 0.26 bis 2.6 µm2 s-1. Dabei ist die Mobilität direkt von der Hydrophilie des Substrates abhängig. Die beobachteten Ergebnisse zeigen deutlich die entscheidende Rolle der Polarität der verwendeten Substratoberflächen auf die Lipidmobilität, die sehr wahrscheinlich mit der Präsenz einer variablen Wasserschicht zusammenhängt. Die untersuchten Polymerkissen eröffnen neue Möglichkeiten für die insitu Modulierung der Charakteristika von Lipidschichten, um funktionale transmembrane Proteine zu integrieren. Aufgrund der unterschiedlichen physiko-chemischen Eigenschaften kann das Verhalten der Lipidschichten und der transmembranen Proteine nach den spezifischen Anforderungen des Modellsystems angepasst werden. Die funktionale Integration wurde am Beispiel des transmembranen Proteins BACE nachempfunden. Die Mobilität des integrierten BACE wurde auf allen Polymerkissen beobachtet. Im Gegensatz dazu wurde auf harten Substraten keine BACE Mobilität gefunden. Die Aktivität des integrierten BACE wurde durch die enzymatische Spaltung eines APP-Analogons nachgewiesen. Bemerkenswerteweise wurde ein Anstieg der BACE Aktivität auf den Polymerkissen um den Faktor 1,5 bis 2,5 im Vergleich zu den auf harten Substraten integrierten BACE beobachtet. Zusammenfassend, die verwendeten Polymerkissen bieten vielfältige Möglichkeiten Lipidschichten mit variierenden Eigenschaften für die Integration von transmembranen Proteinen zu erzeugen.